Magnetic attraction transfer devices for use in solid phase radioimmunoassays and in other assay methods

ABSTRACT

A device for transferring, by magnetic attraction, antigen-antibody adsorbent materials from one reaction mixture to another, and to facilitate an efficient rinsing of these materials, and for transferring adsorbent materials used in other assay methods.

This is a divisional of application Ser. No. 680,506 filed Apr. 26, 1976now abandoned.

BACKGROUND OF THE INVENTION

Radioimmunoassay (RIA) methods generally fall into two categories:liquid and solid phase. Liquid phase methods, which involve immunecomplexes in the form of precipitates suspended in a liquid phase, havebeen widely used for detection and quantitation of protein containingantigens, including hormones and viruses. However, solid phase methodsare enjoying more widespread use, particularly for detecting andquantitating viral antigens and anitbodies. The solid phase may involvesurface areas in test tubes (U.S. Pat. No. 3,867,517), in depressionplates, on beads, or any other convenience device (U.S. Pat. No.3,826,619) whose surfaces are of a composition which will adsorb theantigen or antibody reactants and which are of a size and shape whichwill fit into a gamma counter (see also Journal of ImmunologicalMethods, Vol. 5 (1974) pp. 337-344). Advantages in using solid phase RIAsystems include the capability for efficient rinsing and transferring ofthe solid phase, with the antigen-antibody complexes on its surface,without the requirement for centrifugation-resuspension ofantigen-antibody precipitates required in most liquid phase methods.

Spherical beads are particularly useful as solid phases (see Journal ofClinical Microbiology, August 1975, pp. 130-133) because beads can beobtained with extremely uniform diameters (hence surface areas); theyeach contact the bottom of a container at only one small point, they canbe totally immersed in reagent solutions and, for these reasons, givehighly reproducible results in RIA procedures. Such precision isdifficult to obtain with inner surfaces of test tubes or depressionplate wells, probably because: reactants adsorb to the surfaces of thesevessels to various heights during slight "sloshing" attendant withmovements of the vessels; splashing of reagents sequentially addedduring the test and streaming of reagents down the vessels' sides occurduring their delivery. Therefore, unfortunate variations in the surfaceareas exposed to reagents are inevitable. Immersed beads, on the otherhand, allow high uniformity of surface areas exposed to reagents and,when totally immersed, exposure of this uniform surface is highlyreproducible and results in high RIA precision. As examples of thesuperiority of the bead solid phase system for RIA over tube solid phasesystems, the test for Australia antigen has been heretofore convertedfrom the tube to the bead test, and bead tests are the only onescurrently licensed for use in the United States for detection ofhepatitis B antigen in blood donors' sera.

Processing and transferring beads by the presently used methods,however, leaves much to be desired. The use of foreceps (page 339,Journal of Immunological Methods, supra) to transfer each beadseparately is tedious, time consuming and subject to inadvertent smallvariations inevitable when handling is manual. These deficienciessignificantly limit the usefulness of bead assay methods because: (1)the number of assays which can be done at one time under reasonablyidentical conditions of timing is restricted; (2) small variations inmanual handling, especially between different technicians, limit theprecision with which assays can be accomplished and may be one of theprecision limiting factors in the test; and (3) the cost associated withtedious manual manipulation of many individual beads is substantial.

These problems have been partially solved in a design of a test forhepatitis B antigen by allowing beads to remain in the same depressionplate wells throughout the RIA procedure, merely aspirating reagents andrinsing beads in many wells simultaneously with a mechanicallysophisticated, manifold type rinsing-dispensing device, then dumping thebeads by the force of gravity from the depression plate wells into tubesfor insertion into a gamma counter. There are some objections to thisprocedure, however, when considering optimal test conditions for thehepatitis test and other applications in particular. Performing a RIAbead test completely in one well involves reactions with the well'swalls simultaneously with the bead reaction. In effect, then, the wellwall surface competes with the bead surface for reagents introducedlater in the test. Non-specific adsorption of reactants is usuallyfollowed by a small amount of release of reactants into succeedingreagents added, which is unfortunately magnified quantitatively if thesame well is used throughout the test. Transferring the beads to newwells during the test with facility would obviate this problem.Aspiration of fluids from small volume cul de sacs containing beads withlarge surface areas, followed by addition of small volumes of rinsingfluid nevertheless constitutes a relatively inefficient rinsingprocedure because of the difficulty in aspirating all of the fluid inthe well (a small cul de sac containing an object with a maximum surfacearea, a sphere, hence attracting maximum fluid volume due to capillaryor surface tension effects). Also, this approach does not eliminate thenecessity for transferring beads one-at-a-time by forceps into the wellsinitially. If there are dozens of wells and a short incubation period,treatment of the first and last beads will necessarily be significantlydifferent. Similarly, exact volumes of new reagents must be delivered toeach separate well during the RIA process. Thus, beads exposed firstduring this procedure will not be treated identically to beads exposedat the last of the dispensing period. Therefore, when large numbers ofbeads are employed in an assay run (as when testing many differentspecimens) and short reaction periods are desirable (30 minutes, forexample), uniformity in test conditions is seriously compromised and theprecision of the assay suffers substantially. Inability to expose allbeads to the same reagents simultaneously thus limits the usefulness ofany RIA procedure, but particularly those procedures involving manyspecimens, short incubation times and numerous reagents.

SUMMARY OF THE INVENTION

A process and means for carrying it out are provided in accordance withthe invention wherein magnetic force is used to move antigen-antibodycoated solid phase units from one place to another, i.e., from apre-dispensed reaction mixture to reaction mixture, into and out oflarge volumes of rinsing fluids and, finally, to test tubes or vialswhich are to be inserted into a gamma counter. Coated solid phase unitsusable in other assay methods may likewise be transferred in accordancewith the invention.

It is therefore a principle object of this invention to provideapparatus for use in RIA determinations, or any other chemical reactionin which a solid phase is essential, which allows a high degree ofmechanization and temporal exactness in the transfer of the solid phasefrom one location to another.

Another object of the present invention is to facilitate the processingof large numbers of units simultaneously under extremely uniformconditions, so as to yield highly reproducible results in solid phaseassays with large numbers of specimens.

A further object of this invention is to facilitate extremely efficientwashing of solid phase units to remove contaminating reactants whichdecrease the sensitivity and specificity of solid phase analyticalsystems.

A still further object of this invention is to provide an efficient, yetmechanically simple and trouble-free device for rinsing solid phaseunits, a requirement of all solid phase methods, thus eliminating therequirement for mechanically more sophisticated, and consequently moreexpensive aspiration-rinse devices presently in use.

These and other purposes and advantages will become more apparent fromthe following more detailed description of the invention when taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a greatly enlarged sectional view of one form of thesolid-phase units of the present invention;

FIG. 2 is a greatly enlarged sectional view of a second embodiment ofthe solid-phase unit of the present invention;

FIG. 3 is an expanded sectional view showing part of one form of thetransfer device of the present invention in relation to a testreceptacle tray;

FIG. 4 is a perspective view of a second embodiment of the transferdevice of the present invention;

FIG. 5 is an expanded sectional view of a third embodiment of thetransfer device of the present invention in relation to a testreceptacle tray;

FIG. 6 is a sectional view of a fourth embodiment of the transfer deviceof the present invention;

FIG. 7 is a sectional view of a fifth embodiment of the transfer deviceand test receptacle tray of the present invention;

FIGS. 7A and 7B are respectively bottom plan and top plan views of thetransfer device and tray of FIG. 7; and

FIG. 8 is an expanded sectional view of a sixth embodiment of thetransfer device of the present invention.

Referring to FIG. 1, a solid-phase unit 10 (hereafter referred to as"unit") of the present invention comprises a core 11 of ferrous metalwhen used in the embodiments of FIGS. 3 and 4, or a core 11 of magneticmaterial when used with the embodiment of FIG. 5, for example. The coreis coated with a layer 12 to seal its surface and to prevent chemicalinteractions with the ferrous metal or magnetic core 11, and is furthercoated with an outer layer of an appropriate adsorbent material 13.Layers 12 and 13 may be unitary and the same if composed of a materialthat can act as both a sealant and as an adsorbent. The unit may be ofvarious shapes, sizes and colors, although a sphere with a diameterrange of from about 0.1 mm to about 2.0 cm is preferred because of theincreased surface-to-volume ratio and the ease of handling a pluralityof such units.

Referring to FIG. 2, the second embodiment of a unit 14 of the presentinvention comprises a non-ferrous metal, non-magnetic component 15 ofadsorbent material (or a non-ferrous metal, non-magnetic compound 15coated with a layer of adsorbent material 16) joined in any suitablemanner as by adhesive bonding to a ferrous metal or magnetic component17 coated with a layer 18 to seal its surface and to prevent chemicalinteraction similarly as described with reference to FIG. 1. Unit 14 isused when it is difficult or inconvenient to apply a suitable adsorbentlayer to the ferrous metal or magnetic material.

Any number of different types of undercoatings 12 and 18 can be appliedto the ferrous metal or magnetic material in order to seal the surfaceand thus to prevent unwanted chemical reactions from taking place withthe ferrous metal or magnetic core 11 and 17. Among these coatings areconventional waterproof primer or sealant paints normally used as basecoats for subsequent layers of paint. Required characteristics of thisundercoating are that the coating be waterproof, resistant to rapiddegradation by dilute salt (such as NaCl) solutions, and compatible withthe susequent application of a surface layer of adsorbent material 13.Other efficient coatings 12 and 18, which fulfill these requirements,are a metal coating such as nickel, zinc, chromium, cadmium, copper,gold or any other metal relatively resistant to oxidation or chemicaldegradation by dilute aqueous solutions of salts to thereby effectivelyseal the core from penetration and chemical interaction with variousaqueous salt solutions.

Any number of different types of surface coatings 13 and 16 can beapplied, the choice of coating being dependent upon the type of RIAdetermination to be performed and consequently the nature of thematerials to be adsorbed to the unit. Among these coatings may bevarious plastics (acrylics, polycarbonate, polyethylene, polystyrene,polypropylene, etc.), glass, chromatographic substances or any othermaterial which will adhere to the surface and allow adequate adsorptionof reactants in a particular test system. Plastic surfaces (with orwithout pigments) may be readily applied by dipping or spraying, using asolution of the plastic in an appropriate solvent, and allowing thesolvent to dry. The choice of ssolvents and the method of applicationmust be such as to be compatible with the integrity of layer 12.

The size and shape of the units is not critical except that they shouldbe as uniform as possible in surface area and not be of a designconsisting of deep cul de sacs or which would cause difficulty inuniform exposure of all parts of the unit's surface area to reactionmixtures and washing.

Units can be color-coded (distincitve color coats) or shape-coded(distinctive shapes) so as to allow processing of several differentkinds of units together, in the same test receptacle, and to allowtransfer of several units by a single probe at one time in a mannerwhich will become more apparent hereinafter. In this way, severaldifferent antigen-antibody reactions can be accomplished simultaneouslyin the same test receptacle, with the particular unit identified bycolor or shape. This color coding can be achieved easily by selection ofpigments to be incorporated into sealant coating 12, or by use ofdifferent metal plating having characteristic colors.

A preferred embodiment of unit 10 of FIG. 1 is a ferrous metal sphere0.33 mm in diameter (a Crosman BB) coated with sealant paint, orcadmium, and overlayed with polycarbonate. A preferred embodiment ofunit 14 of FIG. 2 is an imitation pearl 6 mm in diameter bonded withepoxy to a 1/4"×1/4" ceramic magnet coated with sealant paint.

Referring to FIG. 3, a transfer device 19 of the present inventioncomprises at least one magnetic probe 21 capable of transferring atleast one unit which adheres to the probe, and a member 22 for removingthe unit from the probe. Associated with the transfer device is anon-ferrous test receptacle tray 23 having at least one test receptacleor well 24 for each magnetic probe.

A preferred embodiment of the transfer device of FIG. 3 includes a body25 having twenty (only two shown) sixteen-penny common nails 21projecting outwardly thereof as shown. Each nail extends through 3/16"centrally located holes 26 in four 7/8"×5/16 "×1/4" ceramic permanentmagnet discs 27. Each magnet has a 1/8 lb. lift. The four ceramicmagnets 27a are vertically stacked on nail 21a with unlike poles attheir interfaces so as to magnetically attract each other. A set of fourstacked magnets 27b and nail 21b similarly arranged are located adjacentnail 21a and magnets 27a with like poles between the adjacent setsfacing one another so as to magnetically repel each other between sets.(A set of four magnets may have to be removed from the nail, turned 180°and replaced on the nail in order for this magnetic repulsion betweensets to occur.) This process is repeated until all twenty sets of fourmagnets and one nail are in a 4×5 array, thus causing units 10 or 14 tomagnetically adhere to the tips of each of the nails. The nails andtheir surrounding magnets are embedded in body 25 of non-metallicmaterial, such as plastic, or of some suitable non-ferrous material, toa depth that covers the ceramic magnets. Member 22 may be a non-ferrousplate having twenty holes 28 therein through which nails 21 extend.

The FIG. 3 embodiment is used as follows: (1) Device 19 is moved towardtray 23 with probes 21 inserted into test receptacles 24 containingferrous metal units 10 or 14 (not shown) which are to be treated, washedand transferred to reacting mixtures in other like test receptacles 24;(2) Device 19 is then lifted, with single units 10 or 14 adhering toeach probe, and immersed into a bath for washing; (3) The magnetic probedevice 19, with units adhering, is moved directly above another testreceptacle tray 23 so that each well thereof underlies each probe, witheach such well containing an appropriate solution for subsequenttreatment of the units; (4) Member or shield 22 on the magnetic probedevice is shifted relatively away from body 25, thereby causingindividual units to drop into the appropriate wells of thesolution-containing tray; (5) These or other combinations ofmanipulations can be repeated as required in the particular procedurebeing used.

FIG. 4 shows a second embodiment of a transfer device 29 of the presentinvention and shows an electromagnetic probe 31 as typical of severalsuch probes which are embedded in and project outwardly of body 25 (notshown). Each probe 31 is capable of transferring at least one unit 10 or14 which magnetically adheres thereto, and is provided in lieu of probe21. And, associated with this device is a tray 23 having at least onetest receptacle or well 24 for each electromagnetic probe.

A preferred embodiment of the transfer device of FIG. 4 includes a 4×5array of six-penny common nails 31 having fifty turns of enamaledarmature wire 32 wrapped around the top end of each nail. Power issupplied by a battery 33 or by any other source of direct current. Whena switch 34 is closed the nails become electromagnets, each capable ofholding by magnetic force a unit to be treated, washed and transferredto another reaction mixture. Units are released by opening switch 34.The device illustrated in FIG. 4 is otherwise used in a manner similarto that described for the device illustrated in FIG. 3.

FIG. 5 illustrates a third embodiment of a transfer device of thepresent invention. This device 35 includes at least one ferrous metalprobe 36 capable of transferring at least one unit 10 or 14 having amagnetic core which adheres to the probe, and a device 37 to remove theunit from the probe. Associated therewith is a tray 23 having at leastone test receptacle or well 24 for each ferrous metal probe similarly asdescribed in the FIG. 3 arrangement.

A preferred embodiment of transfer device 35 of FIG. 5 includes aplastic body 38 having twenty-four six-penny common finishing nails 36projecting outwardly therefrom in a 4×6 array which are driven through apiece of wood 39 and embedded in body 38. Device 37 includes twenty-fourpairs of 7/8"×5/16"×1/4 rectangular ceramic magnets 40 (the pairs ofmagnets are located in the same configuration as the probes) held inplace by a piece of sheet metal 42, both of which are embedded inplastic body 43, and two L-shaped pieces of metal 44 attached to theplastic body by adhesive bonding.

The embodiment illustrated in FIG. 5 may be used as follows:

(1) Probes 36 of device 35 are moved into wells 24 of tray 23 containingthe specially coated magnetic units 10 or 14 which are to be treated,washed and transferred to other like trays or containers;

(2) Device 35 is lifted with the magnetic units adhering, and immersedinto a bath for washing;

(3) Device 35, with the magnetic units adhering, is moved directly aboveanother tray 23, each well 24 of which contains a solution forsubsequent treatment of the units;

(4) The magnetic base 37 is slid under tray 23 and holds the tray inplace by the two L-shaped pieces of metal 44 which engage opposite endsof the tray;

(5) Device 35 is again lowered until the units on each probe detachwithin the wells by the magnetic attraction of magnets 40;

(6) Device 35 is removed from tray 23 and the units remain in the wellsthereof;

(7) These or other combinations of manipulations can be repeated asrequired in the particular procedure being used.

FIG. 6 shows a fourth embodiment of a transfer device 45 of the presentinvention, which is usable with tray 23. This apparatus includes eitherat least one probe comprising cylinder 46 of magnetic material capableof transferring at least one ferrous metal or magnetic unit 10, 14 whichadheres to the probe, or at least one cylinder 46 of ferrous metalcapable of transferring at least one magnetic unit 10, 14 or anycombination thereof. Associated with this device is at least one testreceptacle for every magnet or ferrous metal probe.

Transfer device 45 of FIG. 6 further includes a body 47 of insulatingmaterial such as plastic having imbedded therein twenty-four 1/4"×1/4"cylindrical ceramic magnets 46 in a 4×6 array. This device, as well asdevices 19 and 35, may be coated with a substance such as epoxy paint 48to prevent adsorption of the reactants to the apparatus and degradationof metallic components by solutions to which they are exposed, and ahandle (not shown) may be added to assist in using any of the describeddevices.

The device shown in FIG. 6 is used as follows:

(1) Device 45 is placed over test receptacle tray 23 containing theunits which are to be treated, washed and transferred to other trays ofsimilar construction;

(2) Transfer device 45 is then lifted away from tray 23 with the unitsadhering to the transfer device;

(3) The transfer device and the adhering units are then immersed into abath for washing;

(4) The transfer device with the units adhering, is moved directly aboveanother tray 23, each well 24 of which contains a solution forsubsequent treatment of the units;

(5) By sliding the transfer device across this tray, the units are wipedoff and fall into the appropriate wells;

(6) These or other combinations of manipulations can be repeated asrequired in the particular procedure being used.

When test receptacle tray 23 is fabricated from a hydrophobic materialsuch as polystyrene, and the well 24 diameter is under 7 mm, thensurface tension is sufficient to retain the fluid in the receptacle evenwhen the tray is inverted or abruptly pushed downward. The inversion orsudden downward push of the tray allows the unit to adhere to the probebut leaves the reaction fluid in the receptacle.

FIG. 7 illustrates a fifth embodiment of a transfer device 49 of thepresent invention. This device is identical to device 45 of FIG. 6except that a wedge 51 is provided on body 47 and a block 52 having aslot 53 is provided on tray 54 which is otherwise identical to tray 23of FIG. 5. Wedge 51 engages within slot 53 on the receptacle tray toallow centering or axial aligning and subsequent attachment of thetransfer device to the test receptacle tray. FIG. 7A is a bottom planview showing a part of device 49, and FIG. 7B is a top plan view showinga part of tray 54. The FIG. 7 device is used in a manner similar to thatdescribed for the transfer device shown in FIG. 6.

FIG. 8 illustrates a sixth embodiment of a transfer device of thepresent invention which includes transfer device 49 and tray 54 of FIG.7. A body 55 of plastic material having at least one funnel-shaped hole56 is provided. There are, of course, as many holes 56 provided as arecylinders 46 in the same configuration thereof. The top of the funnel isas wide or slightly wider than each cylinder 46, while the bottom of thefunnel is as small or slightly smaller than each well 24 of the testreceptacle tray. Body 55 assures that the unit will fall into the propertest receptacle well. Body 55 eliminates the problem of tranfer anddelivery of the units between the transfer device and the testreceptacle tray when the tray has been miniturized. Associated with eachcylinder and funnel is at least one test receptacle well. Also, wedges51 are provided on both device 49 and body 55 for engagement within slot53 of block 52 similarly as in FIG. 7.

The device illustrated in FIG. 8 is used as follows:

(1) Device 45 is placed over test receptacle tray 23 containing thespecially coated units which are to be treated, washed and transferredto other similar trays;

(2) Device 45 is then lifted away from tray 23 with the units adheringto the transfer device;

(3) Device 45 and the units are then immersed into a bath for washing;

(4) Device 45, with units adhering, is placed over body 55 which isdisposed over another test receptacle tray, each well of the traycontaining a solution for subsequent treatment of the units;

(5) By sliding device 45 across body 55, the units are wiped off andfall through funnels 56 and into the appropriate wells;

(6) These and other combinations of manipulations can be repeated asrequired in the particular procedure being used.

Obviously, many modifications and variations of the present inventionare made possible in the light of the above teachings. Also, it shouldbe pointed out that the devices described above may be used in carryingout assay methods employing non-radioactive markers or labels, such asvarious dyes or chemicals, which change colors upon reaction with otherchemicals. It is therefore to be understood that within the scope of theappended claims the invention may be practiced otherwise than asspecifically described.

What is claimed is:
 1. A device for use in solid phase immumoassays, forsimultaneously transferring a plurality of antigen-antibody absorbentunits from one location to another, comprising a transfer element incombination with a tray of non-magnetic material having a plurality ofspaced receptacles therein for containing an appropriate liquid and forcontaining units each comprising a core of ferromagnetic material, eachsaid core being covered with at least one coating for preventingchemical interactions between said core and aqueous chemical solutionsand for absorbing antigens or antibodies on the outer surface thereof,said transfer element having a first portion of ferromagnetic materialoverlying said spaced receptacles in a transfer condition of saidelement, and said element having a second portion of non-magneticmaterial overlying spaces between said receptacles when in said transfercondition, and one of said ferromagnetic core and transfer devicematerials having magnetic properties to effect a magnetic attractionbetween said units and said transfer device, whereby said units areremovable from said receptacles by magnetic attraction while beingmaintained in spaced apart relationship by said non-magnetic materialduring said transfer condition.
 2. The device according to claim 1,wherein cooperating means are provided on said tray and on said transferelement for effecting said aligned condition therebetween.
 3. The deviceaccording to claim 1, wherein said ferromagnetic material of saidtransfer element comprise a plurality of ferromagnetic elementsspatially oriented relative to said spaced receptacles.
 4. The deviceaccording to claims 1, 2 or 3, wherein each said unit has an innercoating for preventing chemical interactions between said core and theaqueous chemical solutions and an exterior coating for adsorbingantigens or antibodies on said outer surface thereof.
 5. The deviceaccording to claim 4, wherein said exterior coating comprises anadsorbing coat selected from the group consisting of polycarbonate,polyethylene, polystyrene, polypropylene, acrylics, glass andchromatographic substances.
 6. The device according to claim 4, whereinsaid inner coating includes a stable metal selected from the groupconsisting of nickel, gold, zinc, chromium, cadmium and copper, foreffectively sealing said core from penetration and chemical interactionwith aqueous chemical solutions.
 7. The device according to claim 6,wherein said exterior coating comprises an adsorbing coat selected fromthe group consisting of polycarbonate, polyethylene, polystyrene,polypropylene, acrylics, glass and chromatographic substances.
 8. Thedevice according to claim 4, wherein said inner coating compriseswaterproof paint material for effectively sealing said core frompenetration and chemical interaction with the aqueous chemicalsolutions.
 9. The device according to claim 8, wherein said exteriorcomprises an adsorbing coat selected from the group consisting ofpolycarbonate, polyethylene polystyrene, polypropylene, acrylics, glassand chromatographic substances.
 10. The device according to claims 1, 2,or 3, wherein said transfer element is at least partially coated with amaterial capable of reducing or preventing adsorption of antibodies orantigens to its surface and of preventing degradation of saidnon-magnetic material of said transfer element during exposure toaqueous chemical solutions used in solid phase immunoassays.
 11. Thedevice according to claim 3, wherein said ferromagnetic elements aredisposed flush with a surface of said transfer element.
 12. The deviceaccording to claim 3, wherein said ferromagnetic elements extendoutwardly of a surface of said transfer element, and a release member ofnon-magnetic material surrounding said ferromagnetic elements isprovided for releasing said units adhering thereto by movement of saidrelease member away from said surface.